Botulism is a neuroparalytic disease that can weaken or paralyze skeletal muscle. The disease is caused by intoxication with one of seven serotypes of botulinum neurotoxin (types A - G). Botulinum neurotoxins (BoNTs) are the most toxic protein toxins of humans and are classified as category A select agents. BoNTs intoxicate neuromuscular junctions through a multistep process involving (a) neuronal cell-binding, (b) internalization into acidic compartments, (c) membrane translocation from acidic compartments, and (d) target recognition and catalytic cleavage of neuronal SNARE proteins required for synaptic vesicle exocytosis. BoNTs are thought to bind to the surface of neurons via a dual receptor mechanism in which the physiologic receptor is a complex composed of gangliosides and protein(s) dual receptors. Using a recombinant receptor binding domain of BoNT, a one-step isolation protocol showed that the BoNT neuronal receptor is a component of a presynaptic receptor complex. The aims of this study will: Aim 1, identify the protein components of the presynaptic BoNT receptor complex. This will be achieved by proteomics and mass spectrometry techniques complemented by immunoprecipitation approaches using BoNT-specific and receptor-specific antibodies. Mm 2 will study the interaction tietween BoNTs and the presynaptic BoNT receptor complex. Utilizing the crystal structures of BoNT receptor binding domains (HCRs), targeted mutagenesis of the HCR domain will be employed to identify the toxin- neuronal receptor interaction sites. Identification of the neuronal receptors for the different serotypes of BoNTs will provide insight and opportunities for the development of novel therapies to inhibit against BoNTs intoxication. Similarly, receptor identification will expand the impact of these studies beyond biodefense, by contributing to improved clinical therapeutic protocols that utilize the BoNTs.